The present invention relates to devices and methods of chemical or biochemical tests that are portable, fast, and easy to use, and in particular, but not exclusively, to tests that can be performed outside of laboratories, and by non-technically-trained operators. More particularly, this invention can be used for in vitro medical diagnostics tests wherein clinical specimens such as blood, saliva, urine, stool, and similar, or other samples such as derived from food, plants, animals, and insects, can be tested at the point-of-care, such as a doctor's or dentist's office, pharmacy, school infirmary, border control, or rural clinic, or in the field such as a farm, food processing plant, restaurant kitchen, or zoo. Other applications include environmental monitoring for microorganisms or detection of bioterrorism agents.
This invention is particularly suited for tests that require sample processing to better enable the detection of analytes. For example, many disease conditions, as well as contamination with infectious agents, are signaled by specific biomarkers specific to the pathogen (e.g., viral or bacterial genes) or the host response to the pathogen (e.g., antibodies against infectious agents). Such biomarkers include proteins, nucleic acids (RNA and DNA), toxins, and metabolites. Methods of detecting such biomarkers include immunoassays for proteins and other large molecules, enzymatic nucleic acid amplification such as polymerase chain reaction (PCR), and a host of other methods that rely on highly-specific binding between proteins and other proteins or biomolecules, or nucleic acid hybridization, in combination with reporters such as dyes and nanoparticles, in optical or electrochemical systems. There has been much technical progress in the miniaturization of detection methods. For instance, polymerase chain reactions for amplification of target nucleic acid sequences can be implemented in small chips. However, direct amplification of crude or raw samples such as blood, saliva, urine, food, drinking water, and environmental samples can severely compromise the viability of such tests, with regard to reproducibility, sensitivity, and specificity. Alternatively, amplification tests work better for samples that are processed prior to amplification. For example, plasma or serum is first separated from whole blood, microorganisms and viruses in the plasma are then lysed to release their nucleic acids in soluble form, the nucleic acids are then isolated in purified, concentrated form for enzymatic amplification. This processing removes substances in the sample that are known to inhibit enzymatic amplification. Also, such processing provides for more consistent amplification, reducing variations due to sample variability. Third, it is important to concentrate nucleic acids that may be sparse in the raw sample. Nucleic acids that have a low concentration in the sample require a relatively large sample volume. On the other hand, most enzymatic amplifications used reaction volumes of 10 to 100 microliters. This comparatively small reaction volume facilitates temperature control and economizes enzymes, with sample volumes of 10 to 30 microliters being common. A concentrated amplification reaction also provides more optical contrast (difference between optical properties of reaction mixture and surrounding background) for optical methods of detection such as fluorescence, luminescence, optical absorption, and also electrochemical methods for detecting the amplification product. However, sample sizes limited to this range, i.e., wherein the unconcentrated sample is added as a component of the total reaction volume, will not contain sufficient amounts of target for reliable amplification and detection. A specific example of considerable medical interest relates to viral load measurements, such as commonly used as a diagnostic for monitoring the progression and treatment of HIV-infected subjects. Viral loads in the range of 1 to 1000 viruses per milliliter of blood need to be measured, thus indicating sample sizes on the range of 100 to 1000 microliters. Ultimately, for the reasons discussed above, the enzymatic reactions for detecting HIV virus be designed for reaction volumes of 10 to 1000 microliters. Accordingly, one object of this invention is a means to concentrate the nucleic acid from samples into a much smaller volume for amplification. As a typical example, the total nucleic acid extracted from 500 microliters of blood can be concentrated in relatively purified form to a volume of 10 microliters (sometimes called an elution in solid-phase extraction methods in general and in nucleic acid purification methods in particular), or can be immobilized on a solid phase such as a porous or fibrous membrane of several to tens of cubic millimeters in volume. Currently, this is done in laboratories with spin columns and centrifuges, but this method is not readily amenable to simple diagnostics devices for use at the point of care.
There is considerable interest in diagnostic devices that integrate all sample processing steps with enzymatic amplification and detection. An objective is to reduce burdening the operator of the test with tasks such as sample processing, preparation of reagents, addition of reagents to the device, sample transfer between steps, and the like, in order to make the test more convenient, reliable, and appropriate for a wider range of applications, especially for use in resource-limited settings. Another desirable feature for POC diagnostics is to avoid or minimize the need for supporting instrumentation such as pumps, temperature controllers, actuators, and similar devices. An autonomous device, that can work independently of an auxiliary equipment would broaden the venues of application of such devices, including home use by consumers.
There has been considerable work over the last twenty years on developing miniaturized fluidic systems (‘microfluidics’) that can process and analyze samples. A useful categorization of microfluidics devices is based on their structure and mode of operation. Many devices are based on plastic structures (‘chip’) in which a network of channels, chambers, manifolds, filters, inlet and outlet ports, and other components are formed. Such devices can perform relatively sophisticated processing and analysis of samples, essentially mimicking the types of operations done on the lab bench. However, these types of devices require means for flow control and fluid actuation. They typically incorporate some types of valves or other flow control devices, and pumping mechanisms for moving fluids through the microfluidic network. Alternatively, these functions can be provided by external instrumentation such as programmable syringe pumps. Such devices tend to be relatively complicated to fabricate and operate, making them inappropriate for many point of care applications.
A second type of device is based on moving fluids by the wicking action of porous materials. The lateral flow strip used in the home pregnancy test is a well-known example. A few drops of sample (e.g., urine or blood) are blotted on one end of a nitrocellulose paper strip, and capillary action moves the sample through zones where various substances or reagents (e.g., antibodies or antigens) are immobilized and can interact with analytes in the sample to produce a detectable signal. In the last decade, there has been much work on generalizing this approach, so-called ‘paper microfluidics’ to realize more complicated assays. In some versions of paper-based microfluidics, channels and flow paths defined by wax printing on filter paper. There are two limitations with paper microfluidics. Firstly, the flow control, especially for processes that require a sequence of fluid operations, is difficult to realize, as paper microfluidic devices do not feature conventional valves or flow-gating devices, such that flow streams can be directed along certain pathways, or flow can be turned on or off. Different flow control features, often of a mechanical nature or mechanism, could be incorporated into such paper-based microfluidic devices to give them more functionality. Such features would add complexity with respect to fabrication and operation, as well as reduced reliability.
A second limitation of paper-based microfluidic devices described in the art is that the volumes of sample and other reagents are comparatively small, often in the 1 to 10 microliter range. This aspect can be distinguished from like plastic chips with channels and reservoirs that can be microfluidic formats scale to large sample volumes. Large volumes will overwhelm the wicking capacity of areas of the paper designated for flow paths. Second, liquid reagents and buffers are not self-contained on the device, or have to be actuated from pouches and other compartments, or added (e.g., by pipetting) at the time of use by the operator.
In summary, it is difficult to simultaneously gain the advantages of plastic chips (e.g., compatibility with large liquid volumes and more sophisticated flow control), and paper microfluidics (e.g., passive fluid actuation, i.e., no active pumping) without incurring limitations inherent in either approach. Two recent examples illustrate this dilemma, Whitesides et al. (U.S. Pat. No. 8,821,810, Sep. 2, 2014) describes a slider chip where a moving member provides or modifies a changeable flow path to implement a sequence of fluid operations. However, Whitesides et at does not provide for a means automatically utilize fluid volumes stored on the chip itself, and describe pipetting steps to add liquid buffer solutions to the chip. Imagilov et al. (US Patent Application Publication US 2013/0309679 A1, Nov. 21, 2013) show a microfluidic device with a sliding component that provides a sequenced operation of fluid transfer steps. However, fluid flow is effected by application of pressure at the inlet of the microfluidic network. In either case, operator intervention and/or connection of the device to supporting devices is then required. For simple diagnostics applications, this falls short of complete automation of “sample in, report out,” without relying on external instrumentation, addition of reagents, dislodging liquids from pouches, or other ‘hands on’ control.
The present invention combines a slider mechanism and wicking action with absorption pads. The slider supports a porous membrane which acts as a binding phase in solid-phase extraction. The slide action provides a temporary fluid connection between a sample well, or a reservoir of liquid prestored on the chip, and an absorbing pad. This structure can be repeated as often as needed to implement more complicated assays. In distinction to prior disclosed approaches, the present invention can be scaled for the large sample and reagent volumes needed for sensitive assays, does not require the addition of liquids after sample introduction, and needs only occasional pushing of a slider, for which timing is not crucial. Also, the approach enables a significant concentration (e.g., from 10 to 1000 times) of the analyte of interest, so that in the detection step, the analyte has been concentrated from a large sample into a much smaller volume.